Diabetes and in particular type 2 diabetes, is now a public health issue in the developed and developing worlds. Costly diabetic complications include cardiovascular disease, retinopathy, neuropathy, and nephropathy. Thus, a significant unmet medical needs exist in both detecting and evaluating patients and screening aging populations of a nation for early onset diabetes. For example, in 2011 it was estimated that 25.8 million children and adults in the U.S. (8.3% of the population), have diabetes. While an estimated 18.8 million have been diagnosed with Type 2 diabetes, approximately 7 million are unaware they have the disease. Based on glycaemic measures from 2005-2008, the Centers for Disease Control and Prevention (CDC), reported that 35% of the U.S. adult population had pre-diabetes, that is an estimated 79 million U.S. adults at risk for developing Type 2 diabetes. Similarly in the UK, in 2014, the number of pre-diabetic is claimed to have risen from 11.6% in 2003 to 35.3% in 2013. That is 17.3 Million of UK adults are estimated to have pre-diabetes. In the U.S. alone, the 2007 total annual cost associated with treatment of diabetics was $174 billion. Thus, preventing and managing diabetes and its complications represents a global public health challenge and is a priority for many National healthcare systems.
Detection of diabetes has been traditionally via measurement of glucose levels in blood and urine. For pre-diabetes a glucose tolerance test is taken whereby, following an oral glucose challenge, blood glucose levels are measured, over a timed period, to monitor the maxima and magnitude before levels are brought back to normal. In addition insulin levels can be measured in parallel however the glucose tolerance test requires admittance as a day patient.
Glycated Hemoglobin (Hb) is formed in a non-enzymatic glycation pathway by which hemoglobin's (Hb) reacts freely with blood plasma glucose, even though it is contained within red blood cells. As red blood cells circulate for about 100-120 days in the body, before their components are recycled by the spleen and liver, measurement of glycated Hb therefore reflects the cumulative exposure to glucose: Normal levels of glucose produce a normal amount of glycated hemoglobin; as the average amount of plasma glucose increases, the fraction of glycated hemoglobin increases. Thus, it is a marker for average blood glucose levels over the months prior to the measurement. Rather than a one off test this provides solid evidence of increasing metabolic problems such as pre diabetes. Indeed measurement of glycated Hb provides a much more reliable measure of how well a patient is controlling their diabetes than food and blood glucose diaries.
Thus, a recently favoured test of glycated Hb is the measurement of HbA1C levels, relative to HbA levels. HbA1C is so named because Hb is usually analyzed and measured by elution from reverse phase high pressure liquid chromatography. The chromatographic separation of total Hb resolves the different types of hemoglobin, principally A and A2, from fetal Hb and disease variant of these two types. A minor resolved elution peak is glycated Hb called HbA1C. The measurement of HbA1C is the preferred test for diabetes and the onset of metabolic disease as glycated Hb is a cumulative and non-patient compliance dependant test. Point of care testing for HbA1c is recommended by the American Diabetes Association, as it is rapid and allows the clinician to address the patient's status immediately, improving patient compliance. Hemoglobin A1C was recently approved for use as a diagnostic tool, and an HbA1C of greater than or equal to 6.5% is the cut-off point for diagnosis. The pre-diabetic state was cited to be an HbA1C>5.7 to 6.4%. The correlation of HbA1C to average glucose concentration was recently validated with patients who have type 2 diabetes mellitus.
However, one diagnostic problem is that HbA1C is not suitable for patients with variant Hb or hemoglobinopathies such as sickle cell trait. This can be because the elution profile of the glycated Hb can be obscured by the elution profile of the variant Hb or it interferes with the differential specificity of HbA1C immunoassays.
A second problem is that, as reported in the WHO 2011 report WHO/NMH/CHP/CPM/11.1, Use of Glycated Hemoglobin (HbA1c) in the Diagnosis of Diabetes Mellitus, current HbA1c assays are “unaffordable in most low and middle-income country settings”.
The invention describes a rapid robust and affordable method for screening population for diabetes and pre diabetic metabolic syndromes by analyzing blood samples.
The invention describes direct mass spectrographic analysis of a blood sample, which can be lysed and optionally diluted 100-1000 fold in water. The Hb species present in the sample can be resolved by direct mass spectral analysis such as matrix assisted laser desorption time of flight mass spectrometry. The Hb α-globin chain resolved from the glycated species—Hb α-globin Glc, and/or Hb β-globin from Hb β-globin Glc can be measured for example by normalized area under the curve or peak height. Variants of Hb and hemoglobinopathies are resolved and are not influential on the measurement of percentage glycated Hb, as the Hb α-chain and/or Hb β-chain and respective glycated (Glc) orthologos can be used as the differential marker of diabetic glycosylation.
The invention describes a method to help manage and reduce a cause of socio-economic burden on a nation, through early detection and monitoring.
The method describes rapid screening of whole blood samples, such as pin prick samples and blood spot cards, subjected to direct mass spectral analysis, such as MALDI-ToF Mass spectrometry. Analysis may be carried out following lysis, for example in distilled deionized water, or by freezing, and optionally massive dilution at the range of 1/10 to 1/8000 (preferably 1/2000) in for example distilled deionized H2O or 0.1% trifluoroacetic acid (TFA) in distilled deionised H2O. The resulting spectra is examined as singly charged ions at the Mass/charge range of 15,000 m/z to 16,200 m/z; and/or the doubly charged ions at 7,550 to 8,100 m/z or 7,550 to 8,200 m/z.                Unglycated α-globin is preferably measured at 7,564 m/z        Glycated α globin is preferably measured at 7,645 m/z        Unglycated β-globin is preferably measured at 7,934 m/z        Glycated β-globin is preferably measured at 8,017 m/z        
The spectra is generated using a matrix, preferably sinapinic acid, and intensity of the characteristic resolved mass peaks of α-globin and glycated α-globin, and/or β-globin and glycated β-globin are measured and a ratio determines the relative percentage glycated globin. The determined relative percentage is indicative of pre-diabetes, diabetes and diabetic patients control of cumulative average blood glucose over the previous 2-3 months.
Thus the invention provides a method of detecting pre-diabetes or diabetes comprising subjecting a blood sample obtained from a subject to direct mass spectral analysis and determining the proportion of glycated hemoglobin (Hb) e.g. glycated α-globin and/or glycated β-globin present in the sample.
“Direct mass spectral analysis” means that the data generated from the mass spectral analysis is used in the method, and not the inferred mass of the components present in the sample.
Pre-diabetes, also referred to as borderline diabetes is usually a precursor to diabetes. It occurs when the blood glucose levels are higher than normal, but not high enough for the patient to be considered to have diabetes. It is often described as the “grey area” between normal blood sugar and diabetic levels. Pre-diabetes may be also be referred to as impaired fasting glucose (IFT), if a patient has higher than normal sugar levels after a period of fasting, or as impaired glucose tolerance (IGT), if a patient has higher than normal sugar levels following eating.
The blood sample can be an untreated sample. Alternatively, the blood sample may be diluted or processed (concentrated, filtered, etc.).
The blood sample can be a whole blood sample collected using conventional phlebotomy methods. For example, the sample can be obtained through venupuncture or as a pin prick sample, such as a finger-stick or heel prick. The blood sample may be a dried blood spot captured on filter paper or other suitable blood spot capture material.
The blood sample is preferably treated to lyse the red blood cells. This can be done by diluting a blood sample in a lysing agent, such as deionized distilled water, preferably at a concentration of 1/1 (i.e. 1 part blood to 1 part lysing agent or distilled deionized water). Alternatively the sample can be frozen to lyse the cells. If the blood sample is a dried blood spot, the blood spot capture material on which the sample is dried can be placed in a lysing agent e.g. distilled deionized water to reconstitute the sample. Alternatively the blood spot can be reconstituted in a suitable buffer prior to lysis.
Preferably the blood sample is diluted preferably after lysis. The blood sample may be diluted 1/10 (i.e. one part sample in 10 parts diluent), 1/500, 1/1000, 1/200, 1/2500, 1/8000 or more. Most preferably the sample is diluted 1/2000 i.e. one part blood sample in 2000 parts diluent. Preferably the diluent is 0.1% trifluoroacetic acid in distilled deionised water, more preferably distilled deionized water.
Preferably the blood sample is not processed between lysis and dilution. In other words the blood sample is only lysed and diluted. Such processing includes concentrating the proteins of interest e.g. Hb, α-globin and/or β-globin; isolating Hb, α-globin and/or β-globin by for example HPLC or treatment with a chemical agent to disrupt or break intramolecular bonds. In particular, the sample is preferably not treated with a reducing agent. More preferably the sample is not treated with dithiothrietol (DTT).
The proportion of glycated α-globin and/or β-globin can be calculated i.e. percentage of α-globin and/or β-globin which is glycated. The percentage is calculated as
            Glycated      ⁢                          ⁢      globin              Total      ⁢                          ⁢      globin      ⁢                          ⁢              (                              Glycated            ⁢                                                  ⁢            globin                    +                      non            ⁢                          -                        ⁢            glycated            ⁢                                                  ⁢            globin                          )              ×  100  ⁢  %
A level of ≥4% glycated α-globin and ≥6% β-globin, is indicative of diabetes. A level of 3-4% glycated α-globin and 4-6% glycated β-globin, is indicative of prediabetes. Preferably the proportion of glycated α-globin in calculated in patients with a hemoglobinopathy or a hemoglobinopathy trait.
Methods of generating mass spectra, such as MALDI-Tof MS, are commonly not quantitative technique. For example the Y axis in these spectra is an indicator of “relative strength” of mass peak within the spectra, but not between mass peaks in one sample versus another sample. In order to overcome this, normalization needs to render Y axis value comparable between sample spectra. Thus the spectra obtained from the direct mass spectral analysis is preferably normalized. The spectra is subjected to data processing which results in a normalized statistically determined index of relative proportion of mass spectra. This converts the qualitative mass spectra into a quantitative value. Normalization is the process of producing a data structure to reduce repetition and inconsistencies of data. Several normalization techniques are possible. Typical normalization methods include percentage of total area at a given point, Square difference and ratio of differences. The percentage difference is calculated asPercentage difference=(Y1-Yref/Y ref×100%)
Wherein Y ref is the minimum Y value of the spectra, and Y1 is Y value for each point.
The square difference is calculated asSquare Difference=(Y1−Y ref)2 
The ratio difference is calculated asRatio Difference=(Ratio1−Ratio 2).
Thus the data from the mass spectra is manipulated in order to provide a quantitative measure of the qualitative change shown on the spectra.
Preferably, the spectral model is created by a method of data processing which results in a normalized statistically determined index of relative proportion of mass spectra within a set range. This renders all spectra comparable such that the median and centile variability at any given mass value can be modelled. Preferably the range is between about 7,000-16,500 m/z, more preferably 7,500-16,200 m/z, most preferably 7,500-8,200 m/z. The single charged and/or double charged molecules of globin can be measured. For the singly charged ions, the spectra at the mass/charge range of 15000 m/z to 16200 m/z is examined. For the doubly charged ions, the spectra at the mass/charge range of 6000 to 8100 m/z, more preferably 7550 to 8100 m/z or 7550 to 8200 m/z is examined.                α-globin is preferably measured at 7564 m/z±5 m/z        Glycated α globin is preferably measured at 7645 m/z±5 m/z        β-globin is preferably measured at 7934 m/z±5 m/z        Glycated β-globin is preferably measured at 8017 m/z±5 m/z        
A normalized statistically determined index of relative proportion of mass spectra within a given range can be calculated from using the total area under the curve of mass spectra. This can then be used to calculate the relative intensity.
The area under the curve of mass spectra is calculated by dividing the mass spectra into a plurality of bins of a given number of m/z. As used herein “Bin” has its usual statistical meaning, for example, of being one of a series of ranges of numerical value into which data are sorted in statistical analysis. For example the bins can be 100 m/z, 50 m/z, 25 m/z, 10 m/z or 5 m/z in size. The smaller the size of the bin used, the more refined the method. Preferably the bin size is 5 m/z.
The relative intensity (Y Axis value) can be calculated by the “square of difference” method and therefore a comparable Y value given for every bin. In this method, the minimum Y value of the spectra (Y ref) was subtracted from the Y value at every bin and the difference was squared. The formula used to calculate square of difference=(y1−yref)2 and the calculated square of difference was then named as “relative intensity”.
The relative intensity at each mass bin in a sample can be captured using commercially available statistical tests such as MATLAB®, Stats Direct™ and Origin 8™. The relative intensity for the mass bins for (i) α-globin and glycated α-globin and/or (ii) β-globin and glycated β globin can be used to calculate the proportion of glycated globin present.
Once the spectra has undergone a method of data processing which results in a normalized statistically determined index of relative proportion of mass spectra, the proportion of glycated globin can be determined by measuring the relative height of the peaks corresponding to the glycated and unglycated globins. For the singly charged ions, the spectra at the mass/charge range of 15000 m/z to 16200 m/z is examined. For the doubly charged ions, the spectra at the mass/charge range of 6000 to 8100 m/z, more preferably 7550 to 8200 m/z is examined.                α-globin is preferably measured at 7564 m/z±10 m/z        Glycated α globin is preferably measured at 7645 m/z±10 m/z        β-globin is preferably measured at 7934 m/z±10 m/z        Glycated β-globin is preferably measured at 8038 m/z±10 m/z        
The analysis of the mass spectra can be easily calculated using a suitable computer software program.
Preferably, the mass spectral analysis carried out is matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS).
Also described is a method of detecting pre-diabetes or diabetes comprising
a) obtaining a blood sample from a subject;
b) subjecting the sample to direct mass spectral analysis;
c) Calculating the proportion of glycated globin present; wherein a percentage glycated α globin ≥4% and ≥6% glycated β-globin is indicative of diabetes, and a percentage glycated globin between 3-4% for α-globin and 3-6% for β-globin is indicative of pre-diabetes. Preferably the percentage of glycated globin is the percentage glycated α-globin, in particular in subjects with a hemoglobinopathy.
In this specification, the verb “comprise” has its normal dictionary meaning, to denote non-exclusive inclusion. That is, use of the word “comprise” (or any of its derivatives) to include one feature or more, does not exclude the possibility of also including further features. The word “preferable” (or any of its derivatives) indicates one feature or more that is preferred but not essential.
All or any of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all or any of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.
Each feature disclosed in this specification (including any accompanying claims, abstract and drawings), may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.
The invention is not restricted to the details of the foregoing embodiment(s). The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.